1) Intracellular signaling activated by collagen-integrin interactions in osteoblastic differentiationWe have previously shown that interaction of type I collagen (COL) with alpha2beta1 integrin causes differentiation in osteoblastic cells. In this study, to clarify how the cell-matrix interaction regulates these phenotypic changes, signaling pathways were examined in murine MC3T3-E1 cells. Attachment of cells to COL stimulated tyrosine phosphorylation of focal adhesion kinase (FAK) and a mitogen-activated protein kinase (MAPK), and enhanced MAPK activity. Inhibition of tyrosine kinase, destruction of focal adhesion, or overexpression of antisense FAK mRNA prevented the activation of MAPK and the increase in alkaline phosphatase (ALP) activity. Transient expression of a MAPK-specific phosphatase also suppressed the elevation of ALP activity. Furthermore, introduction of a constitutively active MAPK kinase enhanced ALP activity in the absence of collagen production. These results demons
… Moretrate that COL-alpha2beta1 integrin interaction facilitates differentiation via the activation of FAK and its downstream signals. These signaling pathways may play an important role in the sequential differentiation of osteoblasts.2) Essential role of matrix-associated BMPs in osteoblastic differentiationIntrinsic mechanisms whereby cells in an osteoblast lineage differentiate into mature osteoblasts are yet unclear. Bone morphogenetic proteins (BMPs) stimulate osteoblastic differentiation and bone formation. We demonstrate that MC3T3-E1 cells constitutively expressed mRNAs for BMP-2 and BMP-4 and accumulated BMPs in collagen-rich extracellular matrices. BMPs associated with the extracellular matrices were involved in the induction of osteoblastic differentiation. MC3T3-E1 cells constitutively expressed type IA and type II BMP receptors. When a kinase-deficient type IA BMP receptor was stably transfected to MC3T3-E1 cells to obliterate BMP-2/4 signaling, these cells not only failed to respond to exogenous BMP-2 but lost their capability of differentiation into osteoblasts. These observations suggest that endogenous BMP-2/4 accumulated in extracellular matrices are essential for the osteoblastic differentiation of cells in the osteoblast lineage. Therefore, the regulatory mechanism of BMP-2/4 actions in osteoblastic cells is a principal issue to be elucidated for better understanding of pathogenesis of osteoporosis.3) Advanced glycation endproducts impair cell-collagen interactions in osteoblastic cellsAdvanced glycation endproducts (AGE) accumulate in matrix with advancing age. Collagen is most susceptible to extensive glycation, and AGE is suggested to be involved in several diseases. AGE inhibited intracellular signaling, such as tyrosine phosphorylation, evoked by the attachment to collagen matrix in osteoblastic cells. Thus, AGE-induced impairment of osteoblastic cells are further to be investigated for the pathogenesis of involutional osteoporosis. Less